DNA methylation is a heritable, epigenetic mark that is conferred by a class of enzymes referred to as the DNA methyltransferases (DNMTs). There are three catalytically active DNMTs: DNMT1 serves as the maintenance methyltransferase during cell division, and DNMT3A and DNMT3B are the de novo methyltransferases that are responsible for establishing methylation patterns early in development. DNMT3L is a cofactor that modulates the activity of the de novo methyltransferases and has important implications in maternal genomic imprinting. DNA methylation and the DNMTs are of particular interest in cancer as aberrant methylation such as gene- specific hypermethylation in tumor suppressor gene promoters and global hypomethylation at repetitive and transposable DNA elements is commonly observed. It is not well understood how aberrant methylation occurs in the cancer genome, and it is the overall goal of this research proposal to determine the unique and overlapping target sites for each of the DNMTs to better understand aberrant regulation of DNA methylation and ultimately develop therapeutic strategies to target altered DNMT activity in cancer. This project will focus on two aims: (1) To define genome-wide DNA methylation and histone modification patterns specific for individual and combinatorial loss of the DNMTs and (2) To identify and characterize unique and overlapping target sites for each DNMT. The first aim will be accomplished by depleting embryonic carcinoma cells (NCCIT) of the different DNMTs both individually and in a combinatorial fashion via siRNA-mediated knockdown. Following depletion of the DNMTs, genome-wide DNA methylation, histone modifications for important marks associated with DNA methylation, and gene expression will be assayed using the methyl-CpG binding domain (MBD) capture technique coupled with massively parallel sequencing (MBD-seq), chromatin immunoprecipitation (ChIP-seq), and RNA microarrays, respectively. These three genome-wide assays will be integrated using bioinformatic data analysis to construct comprehensive epigenomic landscapes representative of each DNMT-depletion scenario. Unique and overlapping target sites of the DNMTs will be determined by aligning the different epigenomic landscapes. Recruitment of DNMTs and temporal epigenetic regulation of known DNMT1 and DNMT3B target sites will be evaluated by utilizing HCT116 1KO (DNMT1 knockout) and 3BKO (DNMT3B knockout) cell lines that have been modified to inducibly express DNMT1 and DNMT3B, respectively in the presence of tetracycline. DNA methylation (bisulfite genomic sequencing), histone modifications (ChIP-quantitative PCR) and gene expression (reverse transcription-quantitative PCR) will be evaluated at 12 hour time points. Finally, this same HCT116 cell line system will be used to characterize and evaluate epigenetic regulation of the identified unique and overlapping target sites of the DNMTs. All together, these experiments will identify target sites for each of the DNMTs as well as provide a better understanding of the epigenetic regulation of these sites in cancer.